Cellular changes after photodynamic therapy on HEp-2 cells using the new ZnPcBr(8) phthalocyanine.

OBJECTIVE: This study investigated the effects of photodynamic therapy (PDT) by using the new photosensitizer Octal-Bromide Zinc Phthalocyanine (ZnPcBr(8)) on the nucleus, mitochondria, and cytoskeleton of HEp-2 cells. BACKGROUND DATA: PDT has been widely used as a therapeutic method for tumor-selective treatment and for other diseases. The therapy requires a photosensitizer, molecular oxygen, and visible light. Different studies have demonstrated that cellular organelles are potential targets for PDT, and the results are dependent on the photosensitizer used in the treatment. In this study, we investigated changes in the nucleus, mitochondria, and cytoskeleton of HEp-2 cells after PDT with the new ZnPcBr(8) phthalocyanine. MATERIAL AND METHODS: HEp-2 cells were cultivated under standard conditions, and then incubated with ZnPcBr(8) (1 micromol/L) for 1 h, and subsequently irradiated with a diode laser light (676 nm, 30 mW, 4.5 J/cm(2)). The cells were further cultured for 1 and 24 h at 37 degrees C in a 5% CO(2) and analyzed with fluorescence microscopy by using specific probes for the investigated organelles. RESULTS: Before PDT, the photosensitizer showed a cytoplasmic diffuse distribution. After PDT, cells showed multinucleation, a punctuated mitochondrial distribution in the perinuclear region, and cellular retraction due to the cytoskeleton changes. All those cellular alterations disrupted homeostasis, contributing to cellular death, which is the major goal of PDT. CONCLUSION: Based on our results and the characteristics of the new ZnPcBr(8) phthalocyanine, mechanistic and biochemical studies must be performed, but it is tempting to consider the chemical as a promising agent for PDT.

Cellular and molecular studies of the initial process of the photodynamic therapy in HEp-2 cells using LED light source and two different photosensitizers.

Photodynamic therapy is an attractive therapeutic procedure for a variety of neoplastic and non-neoplastic disorders that involve a tumor localizing photosensitizer that induces cytotoxicity when activated by a particular wavelength of light. Considering the acceptability of the phthalocyanine photosensitizers, we began to address initial cellular and molecular aspects involved in the therapy that leads to apoptosis or necrosis. After HEp-2 cells had been incubated for 1h with aluminium tetrasulfonate chloride (AlPcS(4)) or zinc phthalocyanines (ZnPc) at 10microM, they were irradiated with an LED prototype (640nm, 70mW, 4.5J/cm(2)) and subsequently analyzed at 0 and 3h after the irradiation through MTT, fluorescence microscopy, Annexin V-PI staining and gene transcription analysis. Based on the results we can address that the two photosensitizers led to 2 different death pathways; for AlPcS(4), the results suggest an initial process of intrinsic death pathway; and for ZnPc, a prostanoid-mediated activation of death machinery is suggested.

Cytotoxicity of octal-bromide zinc phthalocyanine after photodynamic therapy with different light sources.

OBJECTIVE: The objective of this study was to investigate the cytotoxicity of octal-bromide zinc phthalocyanine (ZnPcBr8) at different concentrations (0.25, 0.5, and 1 microM) after irradiating HEp-2 cell cultures with two different light sources: a diode semiconductor laser (660 nm, 30 mW) or an LED (640 nm, 70 mW). In order to obtain comparative results, the irradiation parameters of both light sources were adjusted so that the amount of energy density delivered would be the same (4.5 J/cm2). BACKGROUND DATA: Numerous photosensitizers and light sources used in the treatment of human disease have been studied. Based on these studies, a comparative evaluation of two light sources used in photodynamic therapy (PDT) with ZnPcBr8 was proposed. MATERIALS AND METHODS: HEp-2 cells were incubated with ZnPcBr8 at different concentrations (0.25, 0.5, or 1 microM) for 1 h, irradiated with the diode semiconductor laser (660 nm at 30 mW for 300 sec; 4.5 J/cm2) or the LED laser (640 nm at 70 mW for 128 sec; 4.5 J/cm2), and then incubated in MEM medium for 1 or 24 h. The cells were analyzed using the MTT and trypan blue dye exclusion tests. RESULTS: The results demonstrated that the concentration of 1 microM of ZnPcBr8 was the most effective after PDT administered by both light sources. According to the MTT results, HEp-2-cell viability decreased by 97.96% 1 h after, and by 99.87% 24 h after irradiation with the diode semiconductor laser, and decreased by 94.03% 1 h after, and by 99.21% 24 h after irradiation with the LED. The results obtained using the trypan blue dye exclusion test confirmed the photodynamic efficacy of ZnPcBr8 employed with both light sources. With regard to HEp-2-cell viability, the following results were observed: a decrease of 98.73% 1 h after, and of 99.49% 24 h after irradiation with the diode semiconductor laser; and a decrease of 98.76% 1 h after, and of 99.23% 24 h after irradiation with the LED. CONCLUSIONS: According to our results with the irradiation parameters studied here, both the LED and diode semiconductor laser can be used for PDT in vitro, since both light sources had excellent photodynamic efficacy.

Photodynamic therapy with a new photosensitizing agent.

OBJECTIVE: The aim of this study was to investigate the cytotoxicity of octal-bromide zinc phthalocyanine (ZnPcBr(8)) before and after irradiation with a low-power laser (AsGaAl) and analyze the effects of photodynamic therapy (PDT) on the nucleus of L929 cells. BACKGROUND DATA: One of the most recent and promising applications of phthalocyanine in medicine is in the detection and cure of tumors. We studied the ZnPcBr(8) in agreement with the development of new photosensitizing agents for curing tumors. METHODS: L929 cells were cultivated at standard conditions, incubated with ZnPcBr(8) for 1 h at different concentrations, irradiated with a semiconductor laser, and incubated in MEM medium for 1, 12, or 24 h. Cells were analyzed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) technique and fluorescence microscopy. RESULTS: The results demonstrated that ZnPcBr(8) at 1 microM was the most effective concentration for PDT, with a decrease of 63% after 1 h, 99% after 12 h, and 100% after 24 h in relation to the control group. The fluorescence microscopy results showed that ZnPcBr(8) was localized in the perinuclear region when analyzed 1 h after incubation. Nucleus staining with DAPI made it possible to observe that nuclear fragmentation occurred 24 h after PDT, cytoplasm retraction at 1, 12, and 24 h after PDT, and vacuoles along the cytoplasm at 12 and 24 h after PDT. CONCLUSION: According to the results obtained in this study, L929 cell death caused by PDT with ZnPcBr(8) possesses characteristics of apoptosis mediated by the mitochondria, due to the decrease in cells viability, the subcellular localization, and the photodamage found.

Ultrastructural changes in Tritrichomonas foetus after treatments with AlPcS4 and photodynamic therapy.

The Tritrichomonas foetus is an amitochondrial parasitic protist which causes bovine trichomoniasis, a major sexually transmitted disease in cattle. No effective drugs for this disease have been approved to this date. Photodynamic therapy (PDT) is an experimental treatment that shows great potential for treating bacteria, fungi, yeasts, and viruses. However, the cytotoxic effect of PDT on protozoan has been poorly studied. In this study, PDT with aluminum phthalocyanine tetrasulfonated (AlPcS4) photosensitizer was efficient in killing T. foetus. The mode of cell death in T. foetus after PDT was investigated by transmission electron microscopy. Morphological changes, such as membrane projections, nucleus fragmentation with peripheral masses of heterochromatin, endoplasmic reticulum proliferation, intense cytoplasmic vacuolization, fragmented axostyle-pelta complex, and internalized flagella could be observed. This is the first report to demonstrate cell death in T. foetus after PDT, and thus will open up new lines of investigation to develop new treatments for bovine trichomoniasis.